Radiation treatment is one of the most important cancer-therapeutic approaches. Non-toxic radiosensitizers, used in conjunction with radiation therapy, would enhance the effectiveness of the radiotherapy and, at the same time, reduce the total radiation dose to prevent unwanted side effects. DNA double-strand breaks (DSBs) are the major DNA lesion induced by ionizing radiation (IR) and the dominant cause of radiation sensitivity. It is well established that lack of DSB repair can be lethal in mammalian cells. DNA-dependent protein kinase (DNA-PK) is the key component of the DNA nonhomologous end-joining (NHEJ) pathway that is the predominant pathway for DSB repair in mammalian cells. We have shown that inhibition of DNA-PK kinase activity and/or abrogation of radiation-induced autophosphorylation of DNA-PKcs (the catalytic subunit of DNA-PK) results in severe radiation sensitivity. As the autophosphorylation of DNA-PKcs is a requisite step for NHEJ mediated DSBs repair, molecules that inhibit autophosphorylation without affecting the protein levels of DNA-PK or its enzymatic activity could selectively radiosensitize cells without affecting other cellular processes; whereas molecules that inhibit the kinase activity of DNA-PK also commonly inhibit other PI-3K like protein kinases (ATM and ATR) and cause significant cellular toxicity. In this proposal, we plan to develop both lysate-based and cell-based high throughput screening (HTS) assay to identify chemicals or natural compounds that would either block the autophosphorylation of DNA-PKcs or inhibit the kinase activity of DNA-PKcs. With regard to the approaches, [unreadable] [unreadable]